PROJECT SUMMARY Function and regenerative potential of skeletal muscle decline with age, where loss of skeletal muscle quality is attributed to reduced muscle stem (satellite) cell number and function. Inadequate regenerative response following muscle injuries in an aging population further exacerbate the progression of sarcopenia, and therefore reduced independence and quality of life. Exciting studies using the heterochronic parabiosis model, in which young and aged animals are surgically attached to share circulation, suggest the presence of youthful factors in the circulation can rejuvenate age-acquired deficits in muscle regeneration. Subsequent studies have identified a handful of systemic pro-geronic factors, but discovery of the humoral rejuvenation factors that act on muscle stem cells to restore regenerative function has been relatively more elusive. In this context, emerging evidence suggests that during postnatal growth and development, a period of exceptional regenerative capacity and plasticity are maintained in post-mitotic cells. However, the exact identity and underlying pro-regenerative mechanisms are poorly understood. These juvenile protective factors can be manifested as systemic growth factors or presented as circulating progenitor cells. The working hypothesis of this proposal is that these juvenile pro-regenerative factors decrease with aging and ectopic expression of these factors in circulation will elicit superior rejuvenation effects on aged muscle. Despite tremendous therapeutic promise, the critical barrier that limits the detection of putative anti-geronic factors in circulation is the lack of in vitro tools that recapitulates the dynamic regulation of systemic factors in vivo. Thus, it has been technically challenging to design mechanistic studies and elucidate the function of blood-borne factors on their target tissues. To overcome this challenge, the objective in aim 1 is to engineer a novel 3-dimensional (3D) microfluidic ?parabiosis-on-a-chip? circuit that harnesses the key characteristics of native muscle microenvironment and systemic circulation. By leveraging parabiosis-on-a-chip platform with in vivo validation and proteomics, this innovative system will facilitate detection of novel humoral factors and cells that are responsible for rejuvenation effects in heterochronic parabiosis. As such, aim 2 will address whether exposure to the juvenile systemic milieu in aged muscle by parabiosis confers superior rejuvenation effects both in vitro and in vivo. Successful outcomes from proposed studies will have high and broad implications not only in Geroscience but also in broad scientific fields. Parabiosis-on-a-chip circuit will provide a state-of-the-art pre-clinical testing tool that will facilitate our understanding of the dynamic regulation of circulating humoral factors and can also be utilized as a screening device for drug discovery. More importantly, parabiosis-on-a-chip can be expanded to study other organ systems and translated into human clinical studies by emulating human parabiosis-on-a-chip that blood transfusion cannot recapitulate.